The goal of this project is to determine the mechanism of assembly of the envelope of vesicular stomatitis virus (VSV), the prototype rhabdovirus, which has been widely used to study virus assembly. Like many enveloped viruses, VSV has a matrix (M) protein, which serves as a bridge between the nucleocapsid and the virus envelope. The mechanism of how nucleocapsids and M proteins are assembled at the cytoplasmic surface of host membranes to form virus budding sites is a central question in virus assembly. According to virology textbooks, assembly at the plasma membrane is mediated through a transmembrane network of protein-protein interactions of M protein with nucleocapsids and the envelope glycoprotein (G protein). However, most of the available evidence in the literature argues against this textbook model of virus assembly. The research in this project has developed novel methods of electron microscopy analysis and new biophysical and biochemical tools, which have provided evidence for a new model for assembly of VSV envelopes, as follows: A key step is movement of viral nucleocapsids to membrane-proximal regions of the cytoplasm, where they associate with membrane microdomains that are enriched not in M protein, but rather in G protein. 2) These membrane microdomains serve as sites where association of M protein with nucleocapsids occurs. 3) Importantly, one or more of these steps is regulated by host factors that affect virus assembly. This model will be tested by the following aims: Aim 1 is to test the hypothesis that a key step in the initiation of virus assembly is association of viral nucleocapsids with membrane microdomains that are enriched in G protein. These experiments will use new methods of confocal and electron microscopy analysis to study previously described virus mutants, in which assembly of nucleocapsids into virions is defective. The model will also be tested by complementary biochemical approaches and a modification of the "microdomain proteomics" approach to identify host proteins associated with sites of virus assembly. In Aim 2 the mechanism of assembly of M protein into nucleocapsid-M protein (NCM) complexes will be tested. These experiments will use novel stopped-flow light scattering approaches capable of measuring the rapid rates of these protein-protein interactions. This Aim will also analyze the ability of mutant M proteins to be recruited into NCM complexes in vivo. These experiments will involve complementation of temperature-sensitive M protein mutants, which have been shown previously to be defective in assembly of M protein into virions. These experiments not only provide an in vivo assay for assembly of NCM complexes, but also have the potential to provide important new tools for studying the initiating steps in virus assembly. PUBLIC HEALTH RELEVANCE: Vesicular stomatitis virus (VSV) is an animal virus that has been studied for many years as a prototype for many important human viral pathogens. One of the important areas where study of VSV has yielded novel insights is the question of how viruses assemble their outer membrane (or envelope). Recent technological advances in viral genetics, structural biology, and image analysis of microscopy data have revealed that our "textbook" understanding of VSV envelope assembly is likely to be incorrect. The goal of this project is to test new ideas about how VSV assembles its envelope. This will provide fundamental new information on the molecular basis of how viruses like VSV cause disease, and should contribute to development of novel viruses for use as vaccines and for the treatment of human cancer.